Mechanism of immune transfer by RNA extracts

Summary Immune RNA (I-RNA) was extracted from lymphoid organs of BALB/c mice immunized with AKR lymphoid cells. Incubation of normal BALB/c spleen cells with this I-RNA (but not with normal RNA) resulted in leukocyte migration inhibition reactions (LMIR) against AKR extracts but not against purified protein derivative or BALB/c sarcoma extracts. This transfer was abolished by pretreating I-RNA with RNAse but not with pronase. The active fraction of I-RNA was retained by and could be eluted from Poly-U Sepharose columns. Normal cells pretreated with I-RNA also reacted in the presence of an anti-idiotypic anti-serum of anti-(BALB/c anti-AKR) specificity. Pretreatment of cells with anti-idiotypic serum plus complement did not inhibit the subsequent transfer of LMIR with I-RNA. Idiotypic receptors were expressed on I-RNA treated cells less than one hour after I-RNA treatment. Using an I-RNA of double specificity, the results suggested that I-RNA entered into and acted on the cells through a nonspecific mechanism. Finally, I-RNA could induce BALB/c anti-AKR idiotypic markers in C57B1/6 cells, genetically committed for different idiotypes, while RNA extracted from C57131/6 immune cells could not induce in BALB/ c cells their own genetically acquired idiotypes. This series of data would prove that I-RNA acting as a mRNA is able to induce in normal noncommitted cells the de novo synthesis of antigen receptors similar or identical to those present in the surface of in vivo immunized lymphoid cells of the same strain.     

Prethymic T cell precursors express receptors for antigen

An anti-idiotype serum raised in BALB/c mice against syngeneic lymph node T cells from 2,4-dinitrofluorobenzene (DNFB)-sensitized mice was used to study the early expression of antigen receptors on developing T cells. Normal BALB/c bone marrow cells were treated with either anti-Thy-1.2 plus complement or anti-Thy-1.2 and anti-idiotype plus complement before use in the reconstitution of lethally irradiated syngeneic mice. Five weeks after reconstitution, recipient mice were assayed for both contact sensitivity (CS) and in vitro proliferative responses to DNFB. Mice reconstituted with bone marrow cells treated with both anti-Thy and anti-idiotype sera showed a significant decrease in reactivity to DNFB in both assay systems when compared with mice reconstituted with marrow treated with anti-Thy only. CS response to the noncross-reacting hapten oxazolone was identical in both recipient groups. Bone marrow mixing experiments showed no evidence of anti-idiotype-induced suppressor cells in these experiments. These data provide strong evidence that at least some T cell precursors express receptors for antigen prethymically.     

Idiotype vaccines against human T cell acute lymphoblastic leukemia. I. Generation and characterization of biologically active monoclonal anti-idiotopes

A murine monoclonal anti-tumor antibody termed SN2 (Ab1), isotype IgG1-kappa, that defines a unique human T cell leukemia-associated cell-surface glycoprotein, gp37 (m.w. 37,000), was used to generate monoclonal anti-idiotype antibodies (Ab2) in syngeneic BALB/c mice. The Ab2 were screened on the basis of their binding to the F(ab')2 fragments of SN2 and not to the F(ab')2 of pooled normal BALB/c mice sera IgG1 or to an unrelated BALB/c monoclonal antibody of the same isotype. Fifteen Ab2, obtained from two fusions, were specific for the SN2 idiotope and not against isotype or allotype determinants. To find out whether these Ab2 are directed against the paratope of SN2, the binding of radiolabeled SN2 to leukemic MOLT-4 and JM cells which contain gp37 as a surface constituent was studied in the presence of these anti-idiotopes. Clone 4EA2 inhibited the binding 100% at a concentration of 50 ng and 4DC6 inhibited 90% at a concentration of 250 ng. A third clone 4DD6 gave about 50% inhibition. Similar was the inhibition of SN2 binding to insolubilized MOLT-4 antigen or cell membrane preparation. The binding of SN2 (Ab1) to 4EA2 and 4DC6 was also inhibited by semipurified preparation of gp37 antigen. These results demonstrate that at least two of the anti-idiotope antibodies are binding either at or near the binding site idiotope of SN2. Next, the purified Ab2 was used to immunize syngeneic mice to induce antibody binding to MOLT-4 cells or gp37. Sera from mice immunized with 4EA2 and 4DC6 coupled to keyhole limpet hemocyanin contained antibodies which bind to semipurified gp37 antigen and MOLT-4 cells. Immune sera inhibited the binding of iodinated Ab2 and Ab1 indicating that an anti-anti-idiotopic antibody (Ab3) in mice shares idiotopes with Ab1 (SN2). Also, the binding of iodinated Ab2 to Ab1 was inhibited by rabbit antisera specific for gp37. Collectively, these data suggest that anti-idiotype antibodies 4EA2 and 4DC6 may be useful in the generation of idiotype vaccines against human T cell leukemia.     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Immunization with Leishmania-specific T cell not B cell lines or hybridomas can modulate the response of susceptible mice infected with viable parasites

Monoclonal antibodies (mAb), T cell lines, and T or B cell hybridomas were prepared from BALB/c, CBA, or E1 mice infected with Leishmania mexicana. Various mAb were produced which inhibited the growth and motility of parasites in vitro. T cell lines (hybridomas) were screened for their ability to release interleukin 2 on specific antigen exposure. Passive transfer of mAb or T cell lines to infected adult mice caused little perturbation of parasite growth. Recipient naive mice were immunized with purified Ig or irradiated cells from these sources and were subsequently infected with viable parasites. Only preimmunization with T cell lines (hybridomas) led to exacerbation of parasite growth, although enzyme-linked immunosorbent assays could detect the production of anti-idiotype antibodies in mAb (B cell hybridoma)-immunized mice. Either nylon wool-purified T cells or serum Ig from T cell-immunized mice could be used to immunize further naive recipients for protection against parasite growth. These data have implications for the development of anti-idiotype vaccines for Leishmania antigens.     

Leukocyte integrin-dependent and antibody-independent cytotoxicity of macrophage against allografts

Macrophages (Mphis), but not T cells, infiltrating into the rejection site of either i.p. allografted Meth A (H-2d) fibrosarcoma cells in C57BL/6 (B6) (H-2b) mice or BALB/c (H-2d) skin onto B6 mice are cytotoxic against allografts with H-2d specificity. To determine the mechanisms of specific killing of allografts by allograft-induced Mphi (AIM), we raised approximately 5,000 rat monoclonal antibodies (mAbs) against AIM and selected three of them (R1-73, R2-40 and R1-34), each of which inhibited cytotoxic activity against allografts in a dose-dependent manner. The antigens recognized by R1-73, R2-40 and R1-34 mAbs were defined by immunoprecipitation and Western blot analyses as CD11a, CD18 and CD11b, respectively; and the allografts expressed CD54, a ligand of CD11a or CD11b, suggesting leukocyte integrin-dependent killing. Although Ab-dependent cellular cytotoxicity has been recognized as a mechanism of specific killing by Mphis, the infiltration of AIM into the rejection site of allografts far (approximately 6 days) preceded the appearance of serum IgG Ab specific for the allograft. AIM exhibiting full cytotoxic activity against allografts was also induced in the transplantation site of Fcgamma receptor knockout [(B6x129) F1] mice as well as B10.D2 (H-2 compatible with allograft) and B6-xid (X-linked immunodeficiency with B cell-specific defect) strains of mice. In the latter two strains of mice, the levels of serum IgG Ab to the allograft were negligible. Moreover, the cytotoxic activity of AIM against allografts was not affected by pretreatment of the cells with anti-mouse IgG serum, suggesting Ab-independent cytotoxicity.     

Mouse serum inhibition of cytotoxicity of goat antibodies against mouse thymocytes]

It is shown that the serum of Balb/c, C57Bl/6 and F1(C57Bl/6 x CBA) mice inhibits cytotoxicity of goat antithymocyte antibodies. The addition of the serum into the incubation medium increases the proportion of alive cells from -5% up to 95%. Cytotoxicity was also inhibited by the globulin fraction of the mice serum. It is suggested that normal mice serum contains factors which block cytotoxicity of antibodies against antigen host determinants.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Detection of polymorphism in BALB/c leukemia viruses with mouse antisera.

Antisera produced in mice recognize primarily type-specific antigenic determinants on both the major core protein, p30, and the major envelope proteins, gp70 and p15(E), of the endogenous leukemia viruses (MuLV) of BALB/c mice. Three different mouse sera were investigated in detail. (i) Antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted with the gp70, p15(E), and p30 proteins of MuLV. Certain pools of the C57BL/6 anti-AKR K36 serum contained antibodies which serologically distinguished the p30 proteins of N-ecotropic, B-ecotropic, and xenotropic BALB/c MuLV. (ii) Antisera prepared in BALB/c mice against the BALB/c sarcoma 1315 contained antibodies that reacted with a type-specific antigen of the 1315 MuLV gp70 that is not found on other BALB/c MuLV. (iii) The normal sera of multiparous BALB/c mice contained antibodies that reacted with gp70 and p15(E) proteins of ecotropic MuLV. Sera from some of these mice contained antibodies that serologically distinguished the gp70 of N-ecotropic and B-ecotropic BALB/c viruses. These results emphasize the utility of mouse antisera in the serological typing of MuLV. Furthermore, the antigenic differences observed in the p30 and gp70 proteins should be of particular use in the future analysis of recombinant BALB/c MuLV.     

Regulation of cell-mediated cytotoxicity. III. Synergism of cortico-resistant with anti-thymocyte serum-resistant splenic T cells in the generation of cytotoxic lymphocytes in graft-vs-host reactions.

Lethally irradiated F1 hybrid mice were given an i.v. injection of parental strain spleen cells. Six days later, their spleen cells were used as the effector cells to measure the in vitro cell-mediated cytotoxicity (CMC) of the parental cells. The treatment of the donors with hydrocortisone resulted in a marked decrease of the capacity of their spleen cells to produce a CMC reaction, whereas the treatment with antithymocyte serum (ATS) resulted in an almost complete loss of such activity. The mixing of spleen cells from hydrocortisone-treated parental donors with the spleen cells from ATS-treated parental donors before injection resulted in a synergistic amplification of the cytotoxic response. The anti-Thy-1 serum treatment of either spleen cell population abolished the synergism completely. These results indicate that cortico-resistant T cells act as precursors of cytotoxic lymphocytes and that ATS-resistant T cells produce an amplification of their reaction.     

Antigen-induced murine B cell lymphomas. II. Exploitation of the surface idiotype as tumor specific antigen.

In the accompanying report, we have described the characterization of two unusual murine B cell lymphomas, CH1 and CH2. A heterologous antiserum, which we refer to as "anti-idiotype" serum, has been raised to the detergent-solubilized surface immunoglobulin of CH1. The following criteria have established that this antiserum is specific for the CH1 tumor and that it reacts with V region determinants of the tumor surface IgM: 1) the antiserum reacts with CH1 tumor cells, but not normal mouse lymphoid cells or CH2 tumor cells, in indirect immunofluorescence and C-dependent cytotoxicity testing, 2) capping with the anti-idiotype serum removes all or most of the tumor surface Ig, 3) the antiserum forms a single band of precipitation against serum from CH1 tumor-bearing mice, when tested by double diffusion precipitin analysis, and 4) a single band of precipitation is formed in the electrophoretic migration position of IgM when the anti-idiotype antiserum is tested against serum from CH1 tumor-bearing mice in immunoelectrophoresis. Furthermore, we have demonstrated that this antiserum is useful in monitoring tumor growth and is a potent immunotherapeutic agent. Specifically, 50% of mice injected with a lethal tumor inoculum and given a small dose of anti-idiotype serum 2 days later remain tumor free, whereas all tumor-challenged control mice died within 30 days.     

Regulation of cell-mediated cytotoxicity. II. Synergism of two types of thymus dependent cells in vivo.

Sublethal (500 rads) doses of radiation given to mice before the intravenous injection of allogeneic spleen cells induced the development of an increased cell-mediated cytotoxicity (CMC) of the recipients' spleen cells. The effector cells from the irradiated animals were shown to carry the θ alloantigenic marker and to be capable of transferring adoptive immunity in vivo. On the other hand, irradiation of mice with the same dose before the administration of skin or tumor allografts induced a suppression of CMC. The response of irradiated mice treated with tumor allografts was restored with small numbers of spleen or lymph node cells from syngeneic or semi-allogeneic F₁ hybrid donors. With the use of the appropriate cytotoxic alloantisera, it was demonstrated that the majority of the effector cells generated in the spleens of mice restored with semiallogeneic cells were of host origin. These results demonstrate that the precursors of the cytotoxic lymphocytes are radioresistant and indicate that for their stimulation some radiosensitive T cells are necessary to amplify their reaction to nonlymphoid allografts. Allogeneic lymphoid cells, on the other hand, supply a stimulus which does not require the intervention of such amplifier cells. In this case, irradiation induces a stronger CMC response probably by inactivating radiosensitive cells with suppressor activity.     

Independent regulation of serologically distinct idiotypes in F1 hybrid mice

We have investigated the regulation of expression of two distinct intrastrain cross-reactive idiotypes, CRI_A and CRI_C, characteristic of anti-p-azophenylarsonate (anti-Ar) antibodies of the A/J and BALB/c strains, respectively, in (BALB/c x A/J)F₁ (CAF₁) mice. Such hybrid mice were found to synthesize antibodies with each idiotype when immunized against the Ar hapten group, although the expression of each was significantly reduced as compared with the parental strain. CAF₁ mice were pretreated with idiotypic-specific antibody reagents and subsequently hyperimmunized against the Ar hapten. Analysis of the idiotypes present in immune sera showed that suppression of either CRI did not concomitantly suppress the expression of the other. Alteration of the expression of one idiotype was not, however, without influence on the other; the expression of CRI_C was markedly enhanced in mice suppressed for CRI_A.Abbreviations used in this paper anti-Id(A/J) idiotypic-specific antibodies against A/J serum Ar-specific antibodies - anti-Id(BALB/c) idiotypic-specific antibodies against BALB/c serum Ar-specific antibodies - Ar p-azophenylarsonate - BGG bovine -globulin - BSA bovine serum albumin - CAF₁ F(BALB/c x A/J) - CFA complete Freund's adjuvant - CRIA the major cross-reactive idiotype of A/J Ar-specific antibodies - CRI_C the major cross-reactive idiotype of BALB/c Ar-speck antibodies - CRI_m the minor cross-reactive idiotype of A/J Ar-specific antibodies - DTH delayed-type hypersensitivity - HP hybridoma product(s) - KLH keyhole limpet hemocyanin     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997     

Strain-dependent migration of lymphocytes to the vaginal mucosa after peripheral immunization

We have previously demonstrated a genetic predisposition among mice regarding their ability to be protected against vaginal candidiasis after peripheral immunization. Both BALB/c and (BALB/cx C57BL/6) F1 mice are protected against vaginal candidiasis after subcutaneous immunization with Candida albicans extract and C57BL/6 mice are not protected by this immunization. In the present study, the ability of F1-derived immune cells to transfer protection to naive parental strains was observed in BALB/c recipient mice, but not apparent in B6 recipient mice. This result is highly suggestive that the microenvironment of the B6 mouse is responsible for the susceptible phenotype. Genetic studies using (BALB/cx C57BL/6)F1x C57BL/6 backcross mice demonstrated that two genes appeared to regulate the protective effect of peripheral immunization to vaginal challenge. Microsatellite mapping indicated that candidate loci involved in controlling the immune response to vaginal candidiasis after peripheral immunization included the intercellular adhesion molecule-1 (ICAM-1), the Icam-1 related sequence 1, and the Fc epsilon RII (P<0.01). Thus, the ability of cells to bind to vaginal endothelial cells may play an important role in protection against vaginal candidiasis mediated by peripheral immunization.     

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha)

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.     

Immune response of the mouse to the major core protein (p30) of ecotropic leukemia viruses.

Sera from normal C57BL/6 mice contained low titers of antibodies against proteins of MuLV. Sera from C57BL/6 mice that were immunized with allogeneic leukemia cells sometimes contained high-titered antibodies against the p15 protein of MuLV; these antibodies detected group-specific antigenic determinants of the p15 protein, since reactions were observed with the p15 proteins of both AKR and Moloney viruses. In contrast, antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted strongly with the p30 protein of MuLV, as well as with p15. Antibodies in the C57BL/6 anti-AKR K36 sera detected group-specific antigenic determinants of the p30 protein; reactions were observed with the C57BL/6 anti-AKR K36 serum and the p30 proteins of both AKR and Moloney viruses. It was concluded that mice do have the capacity to respond immunologically to antigenic determinants of the MuLV p30 protein, although in most circumstances this is not observed.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.     

Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor

Repeated immunization of normal C57L/J (H-2b) mice with peripheral T cells from BALB.B (H-2b) mice results in the production of antibodies which react with the T cell receptor. A monoclonal antibody-producing hybridoma, F23.1, was isolated from immunized C57L/J mice showing this property. This monoclonal antibody recognizes approximately 25% of peripheral T cells in BALB mice. It stains approximately the same fraction of T cells and precipitates the same heterodimer as the rat monoclonal antibody described previously that was made against isolated receptor material. The allotypic determinant recognized by this monoclonal antibody is present in most common laboratory strains (BALB, C57BL, CBA, A, DBA, C3H) and is absent in C57L, C57BR, and SJL mice. Sorting peripheral T cells from BALB.B or (SJL X BALB/c)F1 mice for the F23.1+ and F23.1- subsets revealed that both populations contain approximately the same CTL precursor frequency for alloantigen. Thus, the T cell receptor allotype defined by F23.1 is present on CTL. Furthermore, cytotoxicity mediated by an F23.1+ CTL line could be blocked specifically by the F23.1 monoclonal antibody. Under appropriate conditions, the monoclonal antibody F23.1 bound to Sepharose 4B beads can induce resting peripheral T lymphocytes of allotype-positive strains to proliferate.     

Surface expression and partial characterization of an arsonate hapten-specific idiotype-bearing T-cell receptor

Anti-idiotype antibodies raised against the arsonate hapten idiotype have been used to detect arsonate-binding receptors on the surface of peripheral T cells of A/J mice and to isolate this material after biosynthetic labeling for partial chemical characterization. It was found that 2-3% of splenic T cells from arsonate-immune mice specifically bound the hapten using immunofluorescent keyhole limpet hemocyanin as a carrier. In double-immunofluorescence labeling experiments, a high proportion (approximately equal to 70%) of these cells also bound the (Fab')2 fragment of rabbit anti-idiotype antibody in exactly the same patches on the cell as the arsonate hemocyanin antigen. In addition, the anti-idiotype antibody inhibited the binding of the hapten-carrier complex to T cells by approximately equal to 70%. In parallel experiments, fowl antibodies against mouse (Fab')2 fragments bound to 100% of arsonate-binding T cells in the same cell-surface patches as the hapten, and were capable of inhibiting 100% of the hapten-binding cells. Capping, shedding, and resynthesis experiments indicated that the T cells synthesized their antigen-binding idiotype-bearing receptors. Immunoblots of unreduced detergent extracts of purified splenic T cells developed with anti-idiotype antibodies showed bands at 150,000 and 94,000 Da. Equal amounts of protein extracted from liver and analyzed in the same gels as the T-cell material failed to show any reactivity with anti-idiotype antibodies. To confirm the biosynthetic origin of the idiotype-positive materials, detergent extracts from 75Se-methionine- or [3H]leucine-labeled Con A-treated splenic T cells were reacted with anti-idiotype antibodies and the bound material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 2-mercaptoethanol the major band was at 68,000 Da, with variable minor levels of material at 45,000 Da, while when hapten was used to isolate the receptor a dominant 25,000- to 30,000-Da band was seen. We believe that the higher-molecular-weight materials are multimers of the 25,000-30,000 subunit.